( s of up to 8 kbwithout a significant...)High-Titer ,Retroviral Vectors for Gene Delivery

s of up to 8 kb without a significant loss of titer.¹ Because the titers of these pMFG-based minimal vectors are sufficiently high, allowing greater than or equal to 90% of target cells to be infected, the need for antibiotic selection is essentially obviated.

Titer Determination

Fig.3

Coding sequence for the enhanced green fluorescent protein (GFP)⁹ was inserted between the EcoR I and BamH I sites in pFB and pFB-Neo. The 293 cell-based amphotropic retroviral producer cell line HW293-A (unpublished data) was used for virus production. Producer cells were transiently transfected with both of the GFP constructs using the Transfection MBS mammalian transfection kit (Stratagene) with some modifications.⁴ Two days following transfection, viral supernatants were collected, and serial dilutions of each supernatant were used to infect dividing NIH3T3 cells. Two days later, the infected NIH3T3 cell populations were analyzed by fluorescence-activated cell sorting (FACS) (Figure 3). Titers were determined using the highest dilution for which cells containing fluorescence above background are clearly visible in the upper left (UL) quadrant of the plot (1:10⁵ for both viruses). Titers are expressed as the product of the dilution factor multiplied by the percentage of infected cells that fluoresce above background, then multiplied by the number of cells infected. By this definition, pFB-Neo-GFP infected cells showed a titer of 1 x 10⁷ cfu/ml of supernatant, and pFB-GFP gave a titer of 3 x 10⁸ cfu/ml. This experiment was repeated with essentially identical results (data not shown). In addition, for the vector pFB-Neo-GFP, parallel infec
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